Esgro complete plus clonal grade medium

Pluripotent ESGRO Complete Adapted C57BL/6 mouse ESCs, which have been pre-adapted to serum-free and feeder-free culture condition, were obtained from EMD Millipore (Billerica, MA) at passage 12 (with 80% normal male mouse karyotype). The cells were seeded on 0.1% gelatin-coated flasks, and maintained at 37°C in a 5% CO₂ humidified incubator at standard densities (i.e., between 5×10⁴/cm² and 5×10⁵/cm²) in ESGRO Complete Plus Clonal Grade Medium (EMD Millipore). The medium contains leukemia inhibitory factor (LIF), bone morphogenic protein 4 (BMP-4), and a glycogen synthase kinase-3b inhibitor (GSK3b-I) to help maintain pluripotency and self-renewal of the ESCs. Cells were passaged every 2–3 days (when reaching 60% confluence) with ESGRO Complete Accutase (EMD Millipore) at about 1∶6 ratio. C57BL/6 ESCs maintain a stable karyotype under the above passaging condition. The cells used for differentiation and gene expression studies were at passage 18.

Dis3^flox/flox female mice^{10 (link)} were stimulated sequentially with PMCG and hCG and mated to Dis3^flox/flox male mice. Embryos were flushed from the uterus 3.5 days post coitus with pre-warmed ESGRO Complete Basal Medium (Sigma SF002). All wells of a 24-well plate were treated with 0.5 mL Gelatin solution (Sigma-Aldrich SF008) for 30 min at room temperature. After removing Gelatin, 0.5 mL pre-warmed ESGRO Complete Plus Clonal Grade Medium (Sigma SF001) was added into each well of the plate. Embryos at the blastocyst stage were transferred individually into each well. Embryos were cultured in ESGRO Complete Plus Clonal Grade Medium for 5 days at 37^◦C with 5% CO₂ and outgrowth of cells was observed from some embryos. Cells were treated with Accutase (Sigma-Aldrich SF006) at 37^◦C for 1 min to dissociate into single cells. 1–5 single cells were transferred into fresh ESGRO Complete Plus Clonal Grade Medium, cultured, and maintained as a stable cell line. Cells were split when reaching 50% confluence and forming spheroids of 20–50 cells. For differentiation, cells were dissociated, washed with SF002 medium, and cultured in SF002 medium at 37^◦C with 5% CO₂ for 3 days. Transfection of mESCs is described in Method details below.

As described previously [15 ], luciferase was transduced into the 959A2-1 murine iPSCs, which was generated from C57BL/6 (B6) mouse embryonic fibroblasts by introducing Yamanaka factors such as Oct3/4, Sox2, Klf4, and c-Myc without viral vectors. These iPSCs were cultured without serum or feeder cells in ESGRO Complete PLUS Clonal Grade Medium (Millipore, Waltham, MA), differentiated into cardiomyocytes as described previously, and purified on glucose-free medium supplemented with lactic acid [16 (link)] (Fig 1A).

Mouse iPSCs were purchased from System Biosciences (Mountain View, CA, USA). The iPSCs were maintained in ESGRO complete PLUS clonal grade medium (Millipore, Billerica, MA, USA). When the culture reached 80–90% confluence, the cells were passaged at a ratio of 1:4 in 75 cm² tissue culture flasks pre-coated with 0.1% gelatin. Cells of passages 14–18 were used for all the experiments.

Mouse iMuSCs were isolated from the injured TA muscles of C57BL/6J (3–8-week-old female; Jackson Lab, USA) mice four days after laceration injury, while control MuSCs were isolated from uninjured TA muscles. The iMuSCs were separately cultured in ESGRO Complete PLUS Clonal Grade Medium (Millipore, USA) on 12-well tissue culture plates (Corning, USA) for 3 weeks. The medium was then replaced with normal muscle growth medium [Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% Fetal Bovine Serum (FBS), 10% Horse Serum (HS), 1% chicken embryo extract (CEE; Accurate Chemical Co., UK), and 1% Penicillin-Streptomycin antibiotics; unless otherwise mentioned, all from Gibco, USA] and iMuSCs were further cultured and expanded on collagen type IV-coated flasks at 5% CO₂ at 37 °C. C2C12 primary mouse myoblasts (purchased from ATTC, USA) and MuSCs were used as controls and were cultured on collagen type IV-coated flasks in growth medium in 5% CO₂ at 37 °C. Characterization of iMuSCs was performed by applying standard in vitro and in vivo assays.

A murine iPSC line, iPS-MEF-Ng-20D-17 (20D-17) [35 (link)], was provided by Riken BRC through the National BioResource Project of MEXT/AMED, Japan. Murine iPSCs were maintained in an undifferentiated state on 0.1% gelatin-coated tissue culture dishes in the absence of serum and feeder cells using ESGRO Complete PLUS Clonal Grade Medium (Millipore, Billerica, MA). Cells were passaged with Accutase (Millipore) and replated every 3-4 days. A murine lung epithelial type II cell line, MLE12, was purchased from ATCC (Manassas, VA) and cultured in HITES (hydrocortisone, insulin, transferrin, estrogen, and selenium) medium (RPMI 1640, 2% FBS, insulin (5 μg/mL), transferrin (10 μg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), β-estradiol (10 nM), and HEPES (10 nM)). Cells were passaged with Accutase (Millipore) and replated every 3-4 days.